LOCUS       sequence-401410-        6553 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing active Cas13e with VEGFA mRNA targeting gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6553)
  AUTHORS   Kumar S, Hsiao YW, Wong VHY, Aubin D, Wang JH, Lisowski L, Rakoczy 
            EP, Li F, Alarcon-Martinez L, Gonzalez-Cordero A, Bui BV, Liu GS
  TITLE     Characterization of RNA editing and gene therapy with a compact 
            CRISPR-Cas13 in the retina.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Nov 5;121(45):e2408345121. doi: 
            10.1073/pnas.2408345121. Epub 2024 Oct 30.
  PUBMED    39475642
REFERENCE   2  (bases 1 to 6553)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2024-11-5"; volume: "121(45):e2408345121. 
            doi"; pages: " 10.1073/pnas.2408345121. Epub 2024 Oct 30"
COMMENT     Sequence Label: pM149: pAAV.CMV-Cas13e-VEGFA sgRNA
FEATURES             Location/Qualifiers
     source          1..6553
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    126..146
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        147..395
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     147..167
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     318..337
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        588..891
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        892..1095
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1046..1066
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    complement(1117..1141)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1150..1170
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3493..3513
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3520..3546
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(3550..3570)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    3601..3808
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3816..3956
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3816..3945
                     /label=AAV2 ITR
     rep_origin      4031..4486
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4118..4137)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4328..4349
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4503..4522)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4622..4644
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4682..4700)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4768..4872
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4873..4941,4942..5733)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5091..5110)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5904..6492
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6393..6412
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgg gcgacctttg gtcgcccggc
       61 ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctt
      121 ctagacaact ttgtatagaa aagttggagg gcctatttcc catgattcct tcatatttgc
      181 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
      241 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
      301 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
      361 tggctttata tatcttgtgg aaaggacgaa acaccggctg gagcagcccc cgatttgtgg
      421 ggtgattaca gctatgtgct ggccttggtg aggtttgatc cggctggagc agcccccgat
      481 ttgtggggtg attacagctt tttttgaatt ctgatgcggt gaagctttag ttattaatag
      541 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
      601 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
      661 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
      721 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
      781 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
      841 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
      901 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
      961 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
     1021 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
     1081 tatataagca gagctggttt agtgaaccgt cagatcaccc agctttcttg tacaaagtgg
     1141 gccaccatgc caaagaagaa acggaaggtc gctcaggtct ctaagcagac atccaagaag
     1201 agggaacttt ccattgatga atatcaaggt gccagaaaat ggtgttttac aatcgccttt
     1261 aataaggcct tggtcaatcg ggacaagaat gacggacttt tcgttgaatc ccttctccgc
     1321 cacgaaaaat actccaagca tgattggtac gatgaggaca ccagggcatt gatcaaatgt
     1381 agcacacaag ccgcaaatgc taaggcagag gctcttcgaa attacttctc acattacagg
     1441 cacagtccag ggtgcttgac cttcaccgca gaggacgagt tgcggactat tatggagcgg
     1501 gcatacgaaa gggccatatt cgaatgtaga cgacgcgaaa cagaagtcat tattgagttc
     1561 ccctcactgt tcgagggcga taggataact acagctgggg tagtcttttt tgtctctttc
     1621 ttcgttgaaa gacgagtgct cgaccgactc tacggagccg tgagcggtct gaaaaagaat
     1681 gagggccaat acaaactcac ccgaaaggca ctctccatgt actgtcttaa ggattcccga
     1741 tttaccaagg catgggacaa aagggtattg ttgttcaggg atatcctggc acagttgggg
     1801 cgaattcccg ccgaagcata tgaatactac cacggcgaac agggtgataa aaaacgcgct
     1861 aatgataacg agggcacaaa tccaaagcgc cataaagaca aattcattga attcgcattg
     1921 cattatcttg aagcccagca cagcgaaatc tgcttcggta ggcgccacat tgtacgagaa
     1981 gaggctggtg ctggagatga acataagaag caccgcacca aggggaaggt agttgtcgat
     2041 tttagtaaga aagatgaaga ccagtcatat tacatatcca agaataacgt aattgtgcgc
     2101 attgataaga acgccggacc acgctcttat agaatggggc tgaatgagct caagtacttg
     2161 gtcttgctta gcctccaggg aaaaggggat gacgctattg ctaagctcta tcgctataga
     2221 caacacgttg agaatatcct cgacgttgtg aaagttacag ataaggataa tcatgtattc
     2281 ctgcctagat ttgtgttgga acagcacggc attggtagga aggcattcaa gcaaaggatc
     2341 gatggaagag tcaagcatgt gcgcggagtg tgggaaaaaa aaaaagcagc cactaacgag
     2401 atgaccctgc acgaaaaagc tagggatatc cttcagtatg taaacgaaaa ctgtactcgc
     2461 tcctttaatc ctggagaata caatagactg cttgtgtgcc ttgttgggaa ggatgtagag
     2521 aactttcaag ccggccttaa gcgactccag ttggctgaac ggatagatgg acgagtgtat
     2581 agtatattcg cacagacttc tacaatcaat gaaatgcatc aagtcgtttg tgaccaaatt
     2641 ttgaacaggc tttgcaggat cggtgatcaa aagctctatg actatgtcgg gctgggtaag
     2701 aaggatgaaa tagattacaa acagaaggtc gcctggttta aggagcacat ttccatcaga
     2761 cgggggtttt tgcggaagaa attttggtac gattctaaaa aaggatttgc aaagctggtg
     2821 gaagaacatt tggagagcgg aggcgggcag cgcgacgtag gtcttgataa gaaatactac
     2881 cacatagacg caattgggcg gttcgaggga gcaaaccctg ccttgtatga aaccctcgcc
     2941 cgagatagat tgtgcttgat gatggcacaa tattttctgg gttcagtacg gaaggaactt
     3001 ggtaacaaga ttgtttggag caatgacagc atcgaattgc ctgttgaagg gagtgtcggt
     3061 aatgaaaagt ccattgtatt ttccgtgagt gactatggca agttgtatgt tcttgacgat
     3121 gcagaattcc ttgggcgaat ctgcgagtac ttcatgcccc acgaaaaggg taaaatacgc
     3181 tatcatacag tttatgagaa aggctttcga gcatataatg acctgcaaaa gaagtgtgtc
     3241 gaagcagtcc ttgctttcga agaaaaggtc gtaaaagcaa agaagatgtc cgaaaaagag
     3301 ggcgcccatt acattgactt cagagagatc cttgcccaga caatgtgtaa ggaagccgag
     3361 aaaactgctg tgaataaggt aaggagagcc tttttccatc accacctcaa gttcgtcata
     3421 gacgaatttg gattgttttc tgatgtgatg aaaaaatatg ggattgagaa agaatggaag
     3481 tttccagtga agcccaagaa aaaaagaaaa gtatcctcct acccatacga tgttccagat
     3541 tacgcttgac aactttatta tacatagttg gaattcctag agctcgctga tcagcctcga
     3601 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     3661 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     3721 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     3781 gggaagagaa tagcaggcat gctggggagg gccgcaggaa cccctagtga tggagttggc
     3841 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     3901 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     3961 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     4021 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4081 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc
     4141 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4201 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     4261 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     4321 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct
     4381 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa
     4441 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     4501 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     4561 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4621 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     4681 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     4741 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4801 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4861 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     4921 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     4981 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     5041 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     5101 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     5161 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     5221 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5281 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     5341 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     5401 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     5461 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     5521 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     5581 gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     5641 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     5701 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     5761 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     5821 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5881 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5941 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6001 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     6061 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6121 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6181 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6241 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6301 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6361 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6421 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6481 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6541 tttgctcaca tgt
//
